Yersinia Pestis

Background

Pathogenesis

Y pestis causes the bubonic, pneumonic, and septicemic plague. People become infected with the plague whenever they breath in Y. pestis particles, so it is an airborne pathogen. It is also transmitted by fleas. Therefore, proper hygiene to prevent infection by fleas and covering your breath when you cough or sneeze is the best way to prevent the pathogen from spreading.

Location

Plague is still found in Africa, Asia, and South America. However, some cases occasionally show up in the Western US.

Why do people care about it?

Plague is one of the most notorious diseases in human history and was responsible for millions of deaths in the 14^th century. In addition, it has modern applications as a potential bioweapon.

Antibiotics

Amoxicillin, Streptomycin, amikacin, ampicillin, arbekacin, gentamicin, and Doxycycline are all antibiotics that are effective against Y. pestis.

Growing Yersinia Pestis

BSL Level

The BSL of Y. pestis is BSL 2. This means that when working with the microbe, access to the laboratory should be restricted, PPE such as lab coats, gloves, and face shields must be worn, procedures that may cause infection are performed in a biological safety cabinet, and autoclave is available for proper disposal.

Agar

The WHO recommends using blood agar such as sheep blood agar or MacConkey agar in order to grow Y. pestis.

Conditions

Y. pestis is facultatively anaerobic which means that it can switch between an aerobic and anaerobic metabolism depending on the circumstances.

Discovery

Even though Y. Pestis was the pathogen behind the plague in the 14^th century, such a discovery wasn’t made until the late 19^th century by Alexandre Yersin. Yersin was a Swiss microbiologist who discovered what the causative agent of the plague was during an outbreak in Hong Kong in 1894.

Procedure for growth

You must first aseptically inoculate the liquid blood culture bottles with the maximum amount of blood or body fluid according to manufacturer instructions.
Tape the plates shut to prevent inadvertent opening and then incubate at 25-28 degrees Celsius in ambient air (air not contaminated by pollutants) for optimal growth. It should take around 5-7 days for a sufficient amount of cultures to grow on the plate.
Y. Pestis samples are most likely taken from a patient. Depending on where on the body the sample originated from and how the sample was obtained, special procedures may need to be taken in order to properly store and transport it.

Visualization of Yersinia Pestis

Differentiating Yersinia Pestis from other microbes

There are a variety of different biochemical tests that can be performed in order to ensure the microbe selected is indeed Y. pestis:

Descriptions of tests in order of appearance:

MacConkey Agar selects for gram negative rods. This means that in order for an organism to grow onto the plate, it has to fulfill those two criteria. Y. pestis fulfills both of those criteria, therefore it can grow on the agar and is positive for both rod-shape and Gram negative peptidoglycan. http://www.bacteriainphotos.com/yersinia_enterocolitica_MacConkey_agar.html
SIM - Motility Test: Above is a motility test. Y. pestis is not motile, therefore it will have a negative motility test and form a thin precipitate such as the test on the left.

https://www.health.ny.gov/guidance/oph/wadsworth/yersinia_pestis.pdf

SIM - Indole Negative: An indole test is used to determine if an organism can produce indol. If it can, a red precipitate will show such as on the left tube. However, Y. pestis cannot form indol so that results of an indol test must be lake that on the right.

https://microbiologyinfo.com/indole-test-principle-reagents-procedure-result-interpretation-and-limitations/

Lactose Production Negative: Lactose fermentation tests are used to determine if the microbe can produce gas from lactose fermentation. If the liquid appears yellow, then the test is positive, and the organism can ferment lactose. However, since Y. pestis cannot ferment lactose, its tube will appear red.

http://www.uwyo.edu/molb2021/virtual-edge/lab22/exp_22a_mtfm.html

Gelatin Hydrolysis Negative: Gelatin hydrolysis tests are used to determine if an organism can produce gelatinase. An organism is put on some gelatin, and if the gelatin turns to a liquid then that means the organism can produce gelatinase. If it remains a gelatin, then the organism tests negative. Y. pestis cannot produce gelatinase, so it should test negative such as in the tube on the bottom.

https://microbeonline.com/gelatin-hydrolysis-test-principle-procedure-expected-results/

Citrate Test - Negative: In citrate tests, an organism is tested to see if it can utilize citrate as an energy source. If it can, then the agar will start to appear blue. If it cannot, the agar will stay the same color. Y. pestis should have a tube that stays the same color throughout the test since it is citrate negative.

https://microbiologyinfo.com/citrate-utilization-test-principle-media-procedure-and-result/

Shigella flexnari and Shigella boydii both grow in similar conditions. Therefore, it can be easy to confuse those organisms for both grow in similar conditions. Therefore, it can be easy to confuse those organisms for Y. pestis.

Molecular Diagnostics

Y. pestis 16s primers in the 5'-3' direction:

Forward: 5’-GAGTTTGATCCTGGCTCAG-3’

Reverse: 5’-ACGGCTACCTTGTTACGACTT-3’

Melting Temperature of selected 16s primers:

Forward: 51.1 degrees Celsius

Reverse: 52.4 degrees Celsius

GC content of selected primers:

Forward: 53%

Reverse 48%

FASTA file of 16s rRNA sequence:

>NR_025160.1 Yersinia pestis strain NCTC 5923 16S ribosomal RNA

ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCGGGAAGTAGTTTACTACTTTGCCGGCGAGCGGCGGACGGGTGAGTAATGTCTGGGGATCTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATGACCTCGCAAGAGCAAAGTGGGGGACCTTAGGGCCTCACGCCATCGGATGAACCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGGGTTGAGTTTAATACGCTCAATCATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGCGCTTAACGTGGGAACTGCATTTGAAACTGGCAAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCACAGAATTTGGCAGAGATGCTAAAGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCACGTAATGGTGGGAACTCAAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCAGATACAAAGTGAAGCGAACTCGCGAGAGCCAGCGGACCACATAAAGTCTGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGG

References

https://emergency.cdc.gov/agent/plague/faq.asp
https://medlineplus.gov/ency/article/000596.htm#:~:text=It%20can%20still%20be%20found,%2C%20Colorado%2C%20and%20New%20Mexico.
https://www.cdc.gov/plague/faq/index.html#vaccine
https://asm.org/ASM/media/Policy-and-Advocacy/LRN/Y-pestis-fixed-figures.pdf
https://www.cdc.gov/training/QuickLearns/biosafety/
https://onlinelibrary.wiley.com/doi/full/10.1002/9781118960608.gbm01174
Bottone EJ, Bercovier H, Mollaret HH. 2015. Yersinia, p. 1–32. In Bergey’s Manual of Systematics of Archaea and Bacteria. American Cancer Society.
Bockemühl J. 1994. [100 years after the discovery of the plague-causing agent--importance and veneration of Alexandre Yersin in Vietnam today]. Immun Infekt 22:72–75.
Sisinthy S, Bhanu N, Aggarwal R. 2000. Identification of Yersinia pestis as the causative organism of plague in India as determined by 16S rDNA sequencing and RAPD-based genomic fingerprinting. FEMS microbiology letters 189:247–52.

About the Researcher: gabe sostre

3rd year microbiology major interested in practicing medicine someday.
Trilingual in English, Spanish, and Japanese.
Born in Orlando, Florida and have lived in the state ever since.
My hobbies include weightlifting, playing video games, and reading. My favorite video game is Super Mario 64 and my favorite book is The Stranger.
My goals in life are to eventually become a physician, become a polyglot, and visit every continent at least once.
研究を読んでくれてありがとうございます！

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